Gabriel T. Liberatore

Macrophages and Microglia Produce Local Trophic Gradients That Stimulate Axonal Sprouting Toward but Not beyond the Wound Edge

Following injury to the mammalian CNS, axons sprout in the vicinity of the wound margin. Growth then ceases and axons fail to cross the lesion site. In this study, using dopaminergic sprouting in the injured striatum as a model system, we have examined the relationship of periwound sprouting fibers to reactive glia and macrophages. In the first week after injury we find that sprouting fibers form intimate relationships with activated microglia as they traverse toward the wound edge. Once at the wound edge, complicated plexuses of fibers form around individual macrophages. Axons, however, fail to grow further into the interior of the wound despite the presence of many macrophages in this location. We find that the expression of BDNF by activated microglia progressively increases as the wound edge is approached, while GDNF expression by macrophages is highest at the immediate wound margin. In contrast, the expression of both factors is substantially reduced within the macrophage-filled interior of the wound. Our data suggest that periwound sprouting fibers grow toward the wound margin along an increasing trophic gradient generated by progressively microglial and macrophage activation. Once at the wound edge, sprouting ceases over macrophages at the point of maximal neurotrophic factor expression and further axonal growth into the relatively poor trophic environment of the wound core fails to occur.

Inhibition of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression reduces dopaminergic sprouting in the injured striatum

After striatal injury, sprouting dopaminergic fibres grow towards and intimately surround wound macrophages which, together with microglia, express the dopaminergic neurotrophic factors glial cell line‐derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). To evaluate the importance of these endogenously secreted neurotrophic factors in generating striatal peri‐wound dopaminergic sprouting, the peri‐wound expression of BDNF or GDNF was inhibited by intrastriatal infusion of antisense oligonucleotides for 2u2003weeks in mice. Knock‐down of both BDNF and GDNF mRNA and protein levels in the wounded striatum were confirmed by in situ hybridization and enzyme‐linked immunosorbent assay, respectively. Dopamine transporter immunohisto‐chemistry revealed that inhibition of either BDNF or GDNF expression resulted in a marked decrease in the intensity of peri‐wound sprouting. Quantification of this effect using [H3]‐mazindol autoradiography confirmed that peri‐wound sprouting was significantly reduced in mice receiving BDNF or GDNF antisense infusions whilst control infusions of buffered saline or sense oligonucleotides resulted in the pronounced peri‐wound sprouting response normally associated with striatal injury. BDNF and GDNF thus appear to be important neurotrophic factors inducing dopaminergic sprouting after striatal injury.

Vampire Bat Salivary Plasminogen Activator (Desmoteplase) Inhibits Tissue-Type Plasminogen Activator-Induced Potentiation of Excitotoxic Injury

Background and Purpose— In contrast to tissue-type plasminogen activator (tPA), vampire bat (Desmodus rotundus) salivary plasminogen activator (desmoteplase [DSPA]) does not promote excitotoxic injury when injected directly into the brain. We have compared the excitotoxic effects of intravenously delivered tPA and DSPA and determined whether DSPA can antagonize the neurotoxic and calcium enhancing effects of tPA. Methods— The brain striatal region of wild-type c57 Black 6 mice was stereotaxically injected with N-methyl-d-Aspartate (NMDA); 24 hour later, mice received an intravenous injection of tPA or DSPA (10 mg/kg) and lesion size was assessed after 24 hours. Cell death and calcium mobilization studies were performed using cultures of primary murine cortical neurons. Results— NMDA-mediated injury was increased after intravenous administration of tPA, whereas no additional toxicity was seen after administration of DSPA. Unlike DSPA, tPA enhanced NMDA-induced cell death and the NMDA-mediated increase in intracellular calcium levels in vitro. Moreover, the enhancing effects of tPA were blocked by DSPA. Conclusions— Intravenous administration of tPA promotes excitotoxic injury, raising the possibility that leakage of tPA from the vasculature into the parenchyma contributes to brain damage. The lack of such toxicity by DSPA further encourages its use as a thrombolytic agent in the treatment of ischemic stroke.

Expression of brain-derived neurotrophic factor and TrkB neurotrophin receptors after striatal injury in the mouse

Brain-derived neurotrophic factor (BDNF) promotes the survival and differentiation of nigral dopaminergic neurons and supports the activity of dopaminergic cells grafted into the striatum. However, little attention has been given to the physiological role of endogenous BDNF and its receptor TrkB within the nigrostriatal dopamine system. We know that striatal injury is followed by long-term stimulation of dopaminergic activity in the striatum, could BDNF play a role in this phenomenon? One week after physical injury to the striatum of C57/Black mice, just before dopaminergic activation becomes obvious, in situ hybridization on coronal sections through mouse striatum reveals that BDNF mRNA expression increases significantly before returning to basal levels within 1 month. Expression of mRNA for TrkB follows a very different pattern. No change of expression of the full-length and catalytically competent TrkBTK+ receptor is seen. However, expression of the truncated form of the receptor TrkTK-, which lacks the catalytic tyrosine kinase domain, does increase and stays elevated for at least 2 months after injury. When combined with observations of dopaminergic activation after striatal injury and the neuroprotective effects of BDNF introduced into the striatum, our findings suggest that BDNF and TrkBTK- do indeed play a role in dopaminergic regeneration and repair.

Imaging the Ischemic Penumbra with 18F-Fluoromisonidazole in a Rat Model of Ischemic Stroke

Background and Purpose— The ischemic penumbra is a major focus of stroke research. 18F-fluoromisonidazole (18F-FMISO), a positron emission tomography (PET) marker of hypoxic cells, has shown promise as a technique to image the penumbra in humans. Our aim was to delineate the pattern of 18F-FMISO binding in a rat middle cerebral artery transient thread-occlusion model, and correlate this with tissue outcome at 24 hours. We hypothesized that the pattern of 18F-FMISO binding would mimic that seen in humans. Methods— Thirty-eight rats underwent 2 hours transient middle cerebral artery (MCA) occlusion, and then received 18F-FMISO at time points from 0.5 to 22 hours post-MCA occlusion and were killed 2 hours later. Autoradiographic assessment of 18F-FMISO binding and assessment (triphenyltetrazolium chloride) of the area of infarction were performed on tissue slices. Results— Until 1 hour after MCA occlusion, 18F-FMISO binding was increased in the entire MCA territory, with little or no infarction visible. Over the next 5 hours, the pattern of binding evolved to a small rim of intensely binding tissue surrounding the infarct core, which itself showed reduced binding compared with the contralateral hemisphere. By 24 hours, there was minimal accumulation of 18F-FMISO binding and a large area of infarction. Conclusions— The pattern of 18F-FMISO binding rats reproduced the pattern seen in humans, consistent with this tracer being a marker of the ischemic penumbra in both species. This technique may have application in studying the ischemic penumbra in animal models, and correlating this with similar studies in humans.

Expression of glial cell line-derived neurotrophic factor (GDNF) mRNA following mechanical injury to mouse striatum.

ALTHOUGH glial cell line-derived neurotrophic factor (GDNF) expression is low in the adult brain, its administration protects dopaminergic neurons against a range of insults, leading to the suggestion of a role in dopaminergic regeneration. If locally produced GDNF is to fulfil a role in dopaminergic regeneration after injury, it seems reasonable to hypothesize that its expression will increase after mechanical trauma. We have demonstrated that GDNF mRNA expression increases within 6 h of using a wire knife to injure adult mouse striatum. Expression doubles after 1 week and remains elevated for at least 1 month. Most GDNF expression is associated with haemosiderin-containing cells, indicating production by brain macrophages. GDNF production by macrophages may be essential for neural regeneration following CNS trauma.

Sprouting of Dopaminergic Axons after Striatal Injury: Confirmation by Markers Not Dependent on Dopamine Metabolism

Striatal injury increases dopamine metabolism in the nigrostriatal system but it is unclear whether this response is due to increased synthesis and activation of tyrosine hydroxylase within existing dopamine terminals and/or branching and sprouting of new terminals. While monitoring the density of tyrosine hydroxylase immunoreactive fibers suggests that sprouting occurs, this technique alone cannot adequately answer this question since the intensity of staining and thus the visibility of individual fibers are intimately linked to dopaminergic activity. However, by examining axons and their branches using markers that are independent of dopamine metabolism it is possible to determine whether dopaminergic sprouting does in fact take place. One month after using a Scouten wire knife to create a small lesion in the left striatum of normal C57/bl-6 mice, silver staining revealed an increase in the total number of neuronal fibers throughout the injured striatum. This was accompanied by intense staining of tyrosine hydroxylase-positive fibers around the wound and an increased density of striatal fibers labeled with dextran-biotin after injection of this neuronal tracer into the substantia nigra 1 month after striatal surgery and 5 days prior to sacrifice. The increase in tyrosine hydroxylase immunoreactivity confirms previous observations of increased dopaminergic activity after striatal injury. The increases in silver staining and dextran-biotin transport provide independent evidence that this increase in dopaminergic activity occurs because of sprouting of new fibers originating in the substantia nigra.

Inducing Stroke in Aged, Hypertensive, Diabetic Rats:

Animal models of ischemic stroke often neglect comorbidities common in patients. This study shows the feasibility of inducing stroke by 2 h of thread occlusion of the middle cerebral artery in aged (56 week old) spontaneously hypertensive rats (SHRs) with both acute (2 weeks) and chronic (36 weeks) diabetes. After modifying the streptozotocin dosing regimen to ensure that old SHRs survived the induction of diabetes, few died after induction of stroke. Induction of stroke is feasible in rats with multiple comorbidities. Inclusion of such comorbid animals may improve translation from the research laboratory to the clinic.

Dopaminergic responses to striatal damage

The improvements obtained by grafting dopamine-rich tissues into the striatum of patients with Parkinsons disease are generally attributed to production and release of dopamine by the graft. However, it is becoming increasingly clear that grafting also stimulates the host dopaminergic system. We provide evidence in a mouse model of striatal damage that surgical cavitation induces a concerted response from the dopaminergic system with proliferation of striatal presynaptic dopamine uptake sites, increased tyrosine hydroxylase activity, increased concentrations of dopamine, dihydroxyphenylacetic acid and homovanillic acid. The response increases with time and ultimately includes contralateral stimulation of striatal tyrosine hydroxylase activity and elevation of dihydroxyphenylacetic acid and homovanillic acid concentrations. The time course and extent of the host dopaminergic response suggests that it may make a significant contribution to observed clinical improvements after intrastriatal transplantation in human parkinsonism.